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3513

Three strictly anaerobic, methanogenic strains JH1T, JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales , and Methanobrevibacter wolinii SHT was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1T, JH4 and JH8 and M. wolinii SHT were 96.2 and 89.0 % respectively, and DNA–DNA hybridization of the isolates and M. wolinii DSM 11976T showed a 20 % reassociation. Strain JH1T exhibited 92 % DNA–DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5–1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37–40 °C and pH 6.5–7.0. The DNA G+C content of strain JH1T was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter , for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1T ( = KCTC 4102T = JCM 18376T).

Tomato (Lycopersicum esculentum) crops grown in several departments of Honduras and heavily infested with the psyllid Bactericera cockerelli were observed in April of 2012 with plant symptoms suggestive of “Candidatus Liberibacter solanacearum” infection. B. cockerelli is a serious pest of potato, tomato, and other solanaceous plants and a vector of “Ca. L. solanacearum” (1,2,3,4). The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (2,3). Disease incidence ranged from 5 to 50% symptomatic plants per field. Tomato (cv. Pony) plant samples were collected from two psyllid-infested commercial fields in the municipalities of Danli and Comayagua in the departments of El-Paraiso and Comayagua, respectively. Total DNA was extracted from leaf tissues of 50 and 20 symptomatic and asymptomatic plants, respectively, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1,3). The DNA samples were tested for “Ca. L. solanacearum” by PCR with primer pairs specific for 16S rDNA (OA2 and OI2c) and the outer membrane protein gene (OMB 1482f and 2086r) of the bacterium (1,2). Ten (20%) of the 50 symptomatic tomato samples were positive for “Ca. L. solanacearum” using both primer pairs and the remaining samples were negative for the bacterium with both primer sets. None of the 20 asymptomatic plants tested positive for “Ca. L. solanacearum”. Amplicons from DNA of two plant samples (one plant/municipality) with each primer pair were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences from the clones (deposited in GenBank as Accession Nos. KC768321 and KC768322) were identical for both locations and showed 99 to 100% identity to several “Ca. L. solanacearum” sequences in GenBank (e.g., JN848753, JN84856, and HM246509). The OMB consensus sequences from the two tomato plants (deposited in GenBank as KC768329 and KC768330) were 100% identical to OMB sequences of Lso in GenBank (CP002371 and JN48754, respectively). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” associated with tomato crops in Honduras. This bacterium has caused millions of dollars in losses to the tomato industry in the United States, Mexico, and New Zealand (2,3,4). Serious damages to tomato crops due to “Ca. L. solanacearum” could expand throughout Central America, especially in those countries where B. cockerelli occurs. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009. (4) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012.

Based on an earlier survey of putative psyllid vectors of apple proliferation (AP), carried out in 2009 and 2010, Cacopsylla picta (Förster) populations infected with ‘Candidatus Phytoplasma mali’ were detected in at least two commercial apple (Malus domestica Borkh.) orchards in southern Finland (1). To establish the presence of ‘Ca. P. mali’ in apple trees, a survey was conducted in 17 commercial apple orchards in August 2012. Phytosanitary inspectors tracked the source of the ‘Ca. P. mali’ by collecting 33 leaf samples from trees showing probable symptoms. Typical symptoms, including elongated stipules and witches' broom, were rare. Total DNA was extracted from leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and screened for ‘Ca. P. mali’ with real-time PCR (2) and the commercial Apple Proliferation Group – complete PCR reaction kit (Loewe Biochemica GmbH, Sauerlach, Germany). Two samples tested positive and results were confirmed with TaqMan PCR and conventional PCR assays and DNA sequencing in the Food and Environment Research Agency (Fera), in the United Kingdom. One positive sample was taken from an orchard in Lohja, southern Finland, where high ‘Ca. P. mali’ incidence in overwintered C. picta was observed in 2010 (1). ‘Ca. P. mali’ was found in a >40-year-old ‘Red Melba’ tree with witches' broom but without elongated stipule symptoms. The other positive sample was collected from an orchard in the Aland Islands, where the infected ‘Lobo’ tree showed symptoms of elongated stipules. This orchard was not monitored for AP vectors. No small fruit symptoms were noted by inspectors or growers in either of the orchards. The positive samples were further analyzed for subtypes using PCR/RFLP and primers AP13/AP10 (3). The amplicons (776 bp) were sequenced and digested with HincII and BspHI (New England BioLabs Inc., Ipswich, MA) following manufacturer's instructions. Both samples proved to be apple proliferation subtypes AT-1 on the basis of RFLP and the sequenced 776-bp region. Sequences of the 776-bp amplicon of the Lohja and Aland isolates showed 100% and 99% identity, respectively, with sequences of apple proliferation isolates (accession nos. L22217.1 and CU469464.1) in GenBank. Both suspected psyllid vectors of ‘Ca. P. mali’ C. picta and C. melanoneura (Förster) occur in Finland, but their distribution, abundance, and transmission specificity is inadequately documented. The next step to evaluate the risk of spread of apple proliferation in commercial orchards is an extensive survey of the occurrence of Cacopsylla species infected with ‘Ca. P. mali’. References: (1) A. Lemmetty et al. B. Insectol. 64:257, 2011. (2) P. Nikolić et al. Mol. Cell. Probes. 24:303, 2010. (3) W. Jarausch et al. Mol. Cell. Probes 14:17, 2000.

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

A novel halotolerant actinomycete, designated strain BNT52T, was isolated from soil collected from Cihanbeyli Salt Mine in the central Anatolia region of Turkey, and examined using a polyphasic taxonomic approach. The isolate was found to have chemical and morphological properties typical of the genus Amycolatopsis and formed a distinct phyletic line in the 16S rRNA gene tree. Strain BNT52T was most closely related to Amycolatopsis nigrescens CSC17Ta-90T (96.7 %), Amycolatopsis magusensis KT2025T (96.6 %), Amycolatopsis sulphurea DSM 46092T (96.6 %), Amycolatopsis dongchuanensis YIM 75904T (96.5 %), Amycolatopsis ultiminotia RP-AC36T (96.4 %) and Amycolatopsis sacchari DSM 44468T (96.4 %). Sequence similarities with other strains of species of the genus Amycolatopsis were lower than 96.2 %. The isolate grew at 20–37 °C, pH 6–12 and in the presence of 0–10 % (w/v) NaCl. The cell wall of the novel strain contained meso-diaminopimelic acid and arabinose and galactose as the diagnostic sugars. Major fatty acids were iso-C16 : 0 2-OH and iso-C16 : 0. The predominant menaquinone was MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylmethylethanolamine. The genomic DNA G+C content was 68.8 mol%. On the basis of the data from this polyphasic taxonomic study, strain BNT52T represents a novel species within the genus Amycolatopsis for which the name Amycolatopsis cihanbeyliensis sp. nov. is proposed (type strain BNT52T = KCTC 29065T = NRRL B-24886T = DSM 45679T).